CD8+ T lymphocytes are the primary immune effector cells that respond to many intracellular pathogens, including viruses, bacteria and parasites, as well as tumors. To recognize infected or neoplastic cells, CD8+ T cells must first be primed by interacting through their T cell receptor with peptide-MHC (pMHC) complexes displayed on the surface of professional antigen presenting cells (pAPC). This can occur either through natural exposure to the pathogen or tumor antigen or by intentionally introducing protein to the antigen processing machinery of a pAPC, as is done during vaccination. One of the most effective ways to introduce proteins for the purpose of priming a CD8+ T cell response is to express that protein using a live, attenuated vector such as the intracellular bacterium, Listeria monocytogenes. Because Listeria enters the host cell cytosol within minutes after initial infection, proteins secreted by the bacterium are readily accessible to the host cell's antigen processing machinery, making Listeria a particularly effective vector for eliciting CD8+ T cell responses. Therefore, we have been investigating the mechanisms involved in the processing of recombinant Listeria proteins for presentation on MHC class I molecules. Our recent studies have uncovered a striking difference in the way recombinant Listeria antigens are processed and presented when compared to endogenously synthesized proteins. Specifically, we have found that pMHC production from secreted Listeria proteins occurs at the same rate, independent of the cellular half-life of the protein from which it is derived. It is our hypothesis that these differences in processing and presentation reflect real differences in the cellular machinery responsible for the degradation of endogenously synthesized proteins vs. exogenously derived bacterial proteins. The studies we propose in this application will focus on determining what factors contribute these differences in processing. We will do this by 1) determining whether differences in protein half-life and processing efficiency are dependent on the endogenous protein processing machinery of the cell and 2) establishing the role Listeria localization plays in determining the processing pathway followed by secreted recombinant proteins. Our studies should provide us with a more detailed understanding of the mechanisms involved in class I antigen processing and presentation. PUBLIC HEALTH RELEVANCE: These studies should increase our understanding of the mechanisms of antigen processing and presentation of proteins expressed using an intracellular bacterial pathogen as a vector. How proteins are degraded and presented in infected cells can impact the quality and magnitude of the cellular immune response. This knowledge is critical to our understanding of how cellular immunity is elicited to antigenic proteins derived from pathogenic organisms and malignant cells. It is expected that this knowledge will aid in the design of effective vaccines.